1. Field of the Invention
The present invention relates to a microscope for observing multi-photon excitation fluorescence, coherent Raman scattering light, second harmonic wave, and the like.
2. Description of the Related Art
Conventionally, for example, as a microscope aiming at observation of depths in a living, body tissue, for example, a multiphoton excitation fluorescence microscope as shown in the publication of the Japanese unexamined patent application, Toku Kai No. 338405 has been known. For example, as the microscope aiming at observation of a living body tissue, such as collagen which forms a cell membrane, a muscular fiber, and protein sequences, for example, a second higher-harmonic-wave microscope using nano-structural dependence nature of the second harmonic wave as shown in the publication of the Japanese unexamined patent application, Toku Kai No. 2000-310799 has been known.
In molecular biology, it has been much demanded that a living activity of particles in the living body, for example, DNA, amino acid, cell organelles, etc., can be observed. Although it is possible to observe the particle in the living body to some extent in case that a conventional fluorescence microscope or multiphoton excitation fluorescence microscope is used, it is necessary to dye target particles by fluorescence pigment. However, it is not desirable to dye a living body by the fluorescence pigment, since it is foreseen that the living body is affected not a little by such facts that toxicity exists in the fluorescence pigment and free movement of the particles is hindered by the fluorescence pigment, and so on.
In recent years, as a microscope for observing three-dimensional distribution of particles in the living body without dyeing, and for observing the like, a coherent Raman scattering microscope for observing coherent anti-Stokes Raman scattering light, has been proposed, for example, as shown in the following patent documents, Toku Hyou 2002-520612, WO 02/06778A1, and US 2003/0011765.
In the multiphoton excitation fluorescence microscope, this fluorescence is observed by using such phenomenon that a sample colored with fluorescent substance is irradiated by super-short pulse laser having a pulse interval of nanosecond, picosecond, or femtosecond at a wavelength λ, and when the irradiation intensity on the sample becomes large enough, multi-photon absorption where two or more (n) of the photon having the wavelength X occurs simultaneously, and the fluorescent substance which has colored the sample is excited by wavelength of λ/n, and the fluorescence having a wavelength that is a little longer than λ/n is emitted.
In the microscope which observes the second harmonic wave, the second harmonic wave is observed by using such phenomenon that the second harmonic wave having two fold of the frequency (the wavelength becomes a half) of the laser light entered from the sample is emitted when laser light is entered into a sample.
In the coherent Raman scattering microscope, the Raman scattering light is observed by using such phenomenon that when a sample is irradiated by excited light, photon energy of illuminating radiation receives inelastic scattering by molecular vibration of the sample, and scattered light having a wavelength which is shifted by an amount of energy equivalent to the proper frequency of a molecule of substance is produced.
By the way, recent years, in the microscope observation of organisms, there is a tendency that various observations combined by the observation techniques mentioned above is required responding to an area of interest of observation object,
For example, in American Journal of Pathology, (Vol. 159, p. 983), there is a description aiming at observation of discharge of integrin molecules and specialization of collagen, at the time of cell morphogenesis of epidermic cells. For such observation, it is desired that observation is carried out in parallel, or selectively in such ways that for epidermic cells as a specimen, as for the whole cell, observation is carried out by using the multiphoton excitation fluorescence, as for collagen, it is carried out by using the second harmonic wave, and as for integrin molecules it is carried out by using the coherent Raman scattering light.
Further, for example, in Journal of Biological Chemistry, (Vol. 17, p. 15441), there is a description aiming at observation of discharge of lipid molecules from a fat cell at the time of insulin medication in the fat cell. For such observation, it is desired that observation is carried out in parallel, or selectively in such ways that for fat cells as a specimen, receptors are observed by using multiphoton excitation fluorescence, cell membranes are observed by using the second harmonic wave, and lipid molecules are by observed using the coherent Raman scattering light.
Further, for example, in Alcohol Health & Research World, (Vol. 21, p. 107), there is a description aiming at an observation of the potential difference on cell membranes in nerve cells and an observation of amino acid molecules discharge accompanying it. For such observation, it is desired that observation is carried out in parallel, or selectively in such ways that for nerve cells as a specimen, potential sensitive is observed by using the multiphoton excitation fluorescence, cell membranes are observed by using the second harmonic wave, and amino acid molecules as a target is observed by using the coherent Raman scattering light.
Further, for example, in PNAS, (Vol. 99, p. 14801), there is a description aiming at an observation of discharge of a calcium wave and sliding movement of myosin molecules having acting fiber form. For such observation, it is desired that the observation is carried out in parallel, or selectively in such way that for muscle cells as a specimen, calcium sensitive pigment is observed by using the multiphoton excitation fluorescence, actin fibers are observed by using a second harmonic wave myosin molecules are observed by using the coherent Raman scattering light.
Further, for example, in PNAS, (Vol. 100, p. 7075), there is a description aiming at diagnosis of cancer using the quantity of NADH and the degree of collapse of collagen fibers.(the degree of collapse of the collagen fibers are detectable with an intensity ratio of the second harmonic wave and the coherent Raman scattering light.) For such diagnosis, it is desired that observation is carried out in parallel, or selectively in such ways that for a cancer cell as a specimen, NADH is observed by using the multiphoton excitation fluorescence, collagen fibers are observed by using the second harmonic wave, and collagen fibers are observed by using the coherent Raman scattering light.